What’s gut got to do with it? The role of the microbiota and inflammation in the development of adiposity and obesity

Obesity is a complex and heterogeneous disease characterized by increased adiposity, ie, the accumulation of lipids and the growth of adipose tissue. In this mini-review, we explore the important role of the gut microbiota and immune system in the development of adiposity. Dysbiosis of the microbiota leads to increased permeability of the gut barrier and bacterial products in the bloodstream, which triggers metabolic inflammation of adipose tissue, muscle, and liver. Inflammation in these highly metabolic organs exacerbates adiposity and contributes to the development of comorbidities associated with obesity. Studies in animal models that manipulate the microbiota and/or inflammation have shown promise in the treatment of obesity.


Introduction
Obesity is a significant health problem affecting millions of people across the globe. The main diagnostic marker of obesity is an increase in the body mass index (BMI), a calculation involving weight and mass. The increased BMI in obese individuals is caused by an increase in white adipose tissue (WAT) due to hypertrophy and hyperplasia of this important energy-storage organ. This increased adiposity can contribute to several comorbidities, including heart disease, cancer, and type II diabetes mellitus (T2DM).
Obesity is thought to begin with abnormal regulation of satiety and hunger signals to the brain from peripheral tissues (eg, gut, liver and adipose) or within the brain itself [1] . This dysregulation leads to changes in eating habits, energy metabolism, and/or autonomic function. Increased consumption of high-fat foods, in particular, tends to lead to metabolic inflammation and insulin resistance (IR) [2,3] . Not surprisingly, these changes to eating habits affect homeostasis in the gastrointestinal tract (ie, the gut), where a delicate balance exists between a large population of commensal microorganisms and the largest immune cell population of the body. Separating these two groups is a single layer of epithelial cells. A significant disruption of the microbial community, like that seen in obesity, is enough to disrupt this system, leading to the activation of the gut immune cells and subsequent systemic inflammation. In this invited review, we summarize the basic pathway proposed to explain how a change of diet and disruption of the gut microbial community leads to inflammation of metabolic organs and the pathophysiology of obesity.

Gut microbiota and adiposity
The gut plays host to a dynamic community of commensal microorganisms with numbers in the trillions. This diverse microbiome plays a crucial role in human health by modulating several physiological functions and maintaining gut homeostasis [4] . The intestinal mucosa is a semipermeable barrier responsible for nutrient absorption from the dietary products within the lumen and preventing pathogens and toxins in the lumen from entering the bloodstream [5] . There are four key components to this complex system-the gut microbiota, the mucus layer, the epithelial monolayer, and the lamina propria [5] . The last two components house large populations of host immune cells. The host's ability to resist infection by pathogens requires strict regulation of this system maintained via the interaction between the host immune system and the microbiome [6] . In this way, the immune system shapes and regulates the gut microbiota to keep a symbiotic relationship between residents and the host [7] . Disease and other threats to homeostasis arise when this relationship becomes unbalanced (ie, dysbiosis).
Maintenance of an intact and robust gut barrier is essential to proper function and overall health. When the gut microbiome is in a state of dysbiosis, the translocation of bacterial products across the epithelial layer can lead to metabolic endotoxemia resulting from increased intestinal permeability [7,8] . This permeability is thought to be a key driver in chronic inflammation due to the translocation of bacterial products such as lipopolysaccharide (LPS), which triggers the host immune response [9] . Certain microorganisms, such as the mucin-degrading bacterium Akkermansia muciniphila (A. muciniphila), strengthen the barrier through interactions with host cells. A. muciniphila utilizes intestinal epithelial mucins as its major carbon and nitrogen sources, degrading the highly glycosylated proteins to acetate and propionate as metabolic end-products [10] . These metabolites, known as short-chain fatty acids, are taken up by enterocytes and serve as energy sources for host epithelial cell turnover, improve the integrity of the epithelial cell layer, and assist the regulation of gut barrier function [11,12] . Some members of the gut microbiota secrete spherical lipid bilayer structures known as extracellular vesicles (EVs) [8,13] that comprise proteins, lipids, nucleic acids, LPS, and other virulence factors and are thought to be conduits for the transfer of genetic material and proteins from bacteria to host [13,14] . EVs interact directly with the gut epithelium and host immune cells to generate signaling pathways protective to the host [15] . In essence, EVs are the functional units of the gut microbiota responsible for regulating host-pathogen interactions [8] . For example, A. muciniphila-derived EV (AmEV) treatment increased tight junction protein expression in Caco-2 cells and improved intestinal barrier integrity in high-fat diet (HFD)-induced diabetic mice [8] . Additionally, the number of AmEVs was higher in the feces of healthy control mice compared with diabetic mice, and supplementation with AmEVs reduced weight gain and improved glucose tolerance [8] . These studies demonstrate the importance of certain members of the microbiota, such as A. muciniphila in maintaining gut barrier integrity and suggest that the use of these organisms as probiotics may be a viable treatment for metabolic inflammation caused by obesity.
Changes to the composition of the gut microbial community are often an indicator of disease, such as obesity. While the definition of what constitutes a healthy gut microbiome remains undefined, the gut microbiota of healthy (nonobese) individuals is highly diverse [16] . Obesity is often associated with the loss of alpha diversity, the microbial diversity within a functional community, such as the gut microbiome at a given point in time [17,18] (Figure 1). An HFD has also been associated with phylum-level changes in gut microbiota involving the two most dominant phyla, Firmicutes and Bacteroidetes [19] . HFD leads to an increased ratio of Firmicutes (known for their ability to metabolize dietary fiber) to Bacteroidetes [17,20] . The expansion of the relative abundance of Firmicutes at the expense of Bacteroidetes is thought to result in an increased energy harvest and excess energy storage in adipose [17,19] . Prolonged exposure to HFD directly correlates with an increase in Bacillaceae, Clostridiaceae, and other Firmicutes in the gut of obese mice, along with a decrease in Bacteroides and other Bacteroidetes [21] . In addition, HFD leads to an overgrowth of Oscillibacter spp. (a Firmicute) whose presence triggers the release of proinflammatory cytokines from immune cells [22] . However, to say that all Firmicutes induce an obese state would be misleading. For example, Lactobacillus acidophilus, a member of the phylum Firmicutes, improved body and adipose mass, glucose metabolism, and gut barrier function in an HFD model of obesity [23] . Other bacterial phyla also play a role in the maintenance of gut homeostasis, including Verrucomicrobia (to which A. muciniphila belongs) and Actinobacteria. Supplementation with Bifidobacterium sp., a popular probiotic, and member of the Actinobacteria phylum, improves outcomes in obesity models through effects on gut barrier function and inflammation [24,25] . Together, these studies demonstrate how small changes to the microbiota community can have dramatic effects on adiposity.

Gut inflammation and adiposity
Proper host-microbial interactions are essential for a healthy gut. These interactions begin with the intestinal epithelium, a single layer of columnar epithelial cells that separates the massive microbial community from a large immune population. Disruption of this delicate system, such as the dysbiosis mentioned earlier, can lead to loss of gut barrier integrity, endotoxemia (from LPS), and systemic inflammation. Key components of the gut barrier include tight junction proteins, namely zonula occludens-1 (ZO-1) and occludin, antimicrobial peptide-secreting Paneth cells, mucin-secreting goblet cells, and secretory immunoglobulin A [26] . The intestinal epithelium is intended to be a continuous barrier to prevent the translocation of bacterial products from the lumen to the bloodstream, but this barrier can be disturbed by obesity and ingestion of HFD [27] due, at least in part, to decreased expression of ZO-1 and occludin [24] ( Figure 1). Beyond molecular disruption of the gut barrier, HFD can lead to physical changes in the gut. Consumption of HFD decreased colon length, cecal weight, cecal and ileal crypt depth, and the number of goblet cells per crypt in rats, a phenomenon that can affect fermentation, nutrient absorption, and potential signals from the gut to the brain [27] . Once intestinal permeability is increased, or conversely, barrier function is decreased, LPS and other bacterial products can pass through the barrier into the bloodstream [27] . An increased concentration of LPS-binding protein (and therefore LPS) in circulation has been reported in obese human patients [26] . In obesity, the presence of LPS in the bloodstream leads to immune signaling and a persistent lowgrade inflammatory state. Furthermore, a large population of immune cells resides in the gut itself and can respond to these changes.
The gut immune population is normally tolerant of microbiota antigens, but when triggered by dysbiosis, can activate inflammatory pathways. Host epithelial and resident immune cells are continuously surveying bacterial cells along the mucosal surface via toll-like receptors (TLRs). These highly conserved receptors detect pathogen-associated molecular patterns, such as LPS, and subsequently activate an appropriate immune response [28] . Homeostasis in the intestine is kept balanced by interactions between TLR4 and LPS, its main ligand [29] . The TLR4 signaling pathway is considered to be one of the main triggers of the inflammatory response induced by obesity. TLR4 expression increases in response to HFD [2,25] , and the resulting proinflammatory signaling in the intestine leads to the production of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ by local macrophages and T cells [27,[30][31][32] . Increased adiposity is associated with an increase in proinflammatory T cells and macrophages in the gut of humans [32] and mice [25] , and along with increased infiltration of these cells in the gut, increased levels of proinflammatory cytokines were found in both species. In contrast, anti-inflammatory cells, such as regulatory T cells (Treg), and their products, such as IL-10, tend to decrease in the obese state [27,31] . Treatment of mice on HFD with a gut-specific anti-inflammatory agent ameliorated many aspects of excessive adiposity, pointing to a key role for gut inflammation in the development of obesity and comorbidities such as IR [31] . Together, these studies point to a subclinical level of inflammation that perpetuates the weakened gut barrier, endotoxemia, and systemic inflammation.

Metabolic inflammation and adiposity
Systemic inflammation activated by dysbiosis and a weakened gut barrier leads to inflammation in metabolic tissues (ie, metabolic inflammation). The most predominant tissue involved in metabolic inflammation is WAT. WAT is organized into several depots throughout the body, including multiple visceral and subcutaneous depots [33] . Adipocytes were long characterized as only a fat storage location, but we now know they play an important role in metabolism as well as endocrine and immune function. They are capable of releasing microRNAs, lipids, and adipokines, the two most significant of the latter being lectin and adiponectin [34] . Leptin controls satiety, and adiponectin regulates insulin sensitivity, and both have been studied in depth related to obesity. Leptin levels in the blood are directly proportional to the mass of WAT, and obesity leads to high levels of leptin in the systemic circulation. In addition to hormone production, adipocytes work in tandem with the stromal vascular faction (SVF), which contains a number of immune cells, including macrophages, B cells, and T cells [35] , to maintain homeostasis in WAT.
During obesity, adipocytes, particularly in the visceral WAT depots, grow in size due to excess calorie consumption. The enlargement of adipocytes leads to changes in tissue structure and increased stress signaling (reviewed in [33] ), the latter of which triggers the production of proinflammatory cytokines and chemokines including, IL-1β, IL-6, IL-8, IL-12, macrophage chemoattractant protein-1, and TNF-α [36][37][38] . Macrophages play a central role in the WAT inflammation seen in obesity. Macrophages of the SVF create so-called "crown-like structures" in WAT. The macrophages in these structures appear to play a role in lipid metabolism but can also promote WAT inflammation in obesity through proinflammatory signaling [39] . The number of macrophages in WAT is positively correlated with adipocyte size and body mass, and these macrophages produce large amounts of IL-6 and TNF-α [37] . TNF-α signaling in WAT has been shown to directly block insulin signaling [38] . Other innate immune cells, including mast cells [40] , innate lymphoid cells (reviewed in [41] ), and natural killer cells [42] , also appear to play a role in WAT inflammation during obesity.
Adaptive immune cells also play a critical role in the development of WAT inflammation during adiposity. Cytotoxic T cells accumulate in the visceral WAT of mice fed HFD and contribute to inflammation, macrophage recruitment, and IR. T Helper (T H ) cells coordinate many of the inflammatory processes during obesity. The number of T cell receptor αβ+ T cells in WAT correlates in a positive fashion with WAT mass [42] . T H cells guide the differentiation of WAT macrophage populations through cytokine production [43,44] . The direction of changes to specific T H subsets in WAT during obesity is less clear and seems to differ between models and experiments. For example, T H 1 cells tend to be positively associated with adiposity [44,45] , while T H 2 cells have been shown to increase [46] or decrease [43] , depending on the experimental conditions and timing of observations. Cytokines from T H 2 cells can polarize macrophages toward the more anti-inflammatory (alternatively activated) phenotype. Treg, potent anti-inflammatory cells, are generally associated with the lean phenotype [45] but have been shown to increase in obesity, most likely as a compensatory mechanism to metabolic inflammation [46] .
WAT inflammation contributes to IR, which can lead to T2DM. IR is due to endocrine effects, not only in WAT but in other highly metabolically active tissues such as skeletal muscle and liver. Although not as well studied as WAT, skeletal muscle does experience some level of inflammation during obesity and contributes to metabolic inflammation through the secretion of myokines (reviewed in [47] ). This inflammation is due to increased numbers of macrophages and T cells entering the tissue and the release of proinflammatory cytokines, such as IL-6 and TNF-α. Macrophages and their production of TNFα, in particular, contribute to atrophy (ie, sarcopenic obesity) seen in many cases of obesity [48,49] . Proinflammatory cytokines and other factors released by hypertrophic WAT can lead to atrophy and inflammation, subsequent to the production of IL-1β and IL-6 in skeletal muscle [50,51] . The deposition of lipids in muscle [52][53][54] along with proinflammatory cytokine production [55,56] contributes to whole-body IR. As with the other tissues discussed thus far, anti-inflammatory IL-10 can reverse or block these negative effects [55,57] . Skeletal muscle is responsible for 80% of glucose absorption under normal conditions; thus, resistance to insulin signaling in this tissue can greatly affect the function of skeletal muscle and promote IR and T2DM systemically [47,58] . The effects of obesity are not limited to skeletal muscle. Smooth muscle myocytes secrete many cytokines and other molecules, such as IL-6, TNF-α, and myonectin, related to the inflammatory process and can become inflamed in obesity, leading to increased muscle inflammation [47] .
Obesity is strongly associated with nonalcoholic fatty liver disease (NAFLD), where the accumulation of ectopic fat causes injury and inflammation to the liver. IR causes triglycerides to build up in hepatocytes, which contributes to NAFLD and increases the potential to develop cirrhosis and/or hepatocellular carcinoma [59] . Cytotoxic T cells and macrophages accumulate in the liver during NAFLD, leading to chronic inflammation [60] . Additionally, levels of inflammatory cytokines, including IL-6, IL-12, IFN-γ, and TNF-α, increase in the liver of obese mice regardless of disease state [61,62] . Increases in TNF-α (and other cytokines that promote TNF-α) promote LPS-induced liver injury [62] . TNF-α is increased in NAFLD and has been found to mediate the early stages of NAFLD as well as the transition to end-stage liver disease [59,63] . The production of TNF-α early in the disease induces the production of other cytokines, such as IL-6, in the area, causing increased inflammation and infiltration of immune cells [63] . IL-6 is markedly increased in patients with severe NAFLD compared with healthy patients [64] and contributes to the development of obesity-related tumors [61] . Furthermore, IL-6 alters the liver's response to insulin, leading to IR [55] . The chronic inflammation in the liver in obese subjects leads to fibrosis and eventually cirrhosis [2,60,62,65] . The eventual loss of liver function leads to significant health impacts.

Summary
The etiology of adiposity and obesity involves many factors, principle among these are dysbiosis and metabolic inflammation. Thus, manipulations of these physiological parameters have long been suggested as potential treatments for obesity. The use of prebiotics [66,67] and probiotics [23,25,68,69] to stabilize the gut microbiota has demonstrated reasonable success in ameliorating or preventing adiposity and certain comorbidities. In humans, the use of probiotics as a treatment for obesity and metabolic disease has shown positive results, although higher doses and longer duration of treatment (compared with rodent models) seem necessary for the best effects [68,[70][71][72] . Probiotics are likely not a "silver bullet", but can be an effective complementary approach to other treatments [72] . Similarly, the use of anti-inflammatory drugs, either systemic [73][74][75] or gut-specific [31] , can also limit fattening, IR, and other effects of metabolic inflammation in laboratory models. Clinical studies in humans are less abundant but do reveal some positive anti-inflammatory effects in WAT [75] . It is possible that a combination of these treatments may be more effective in treating adiposity and metabolic disease.
Many inbred laboratory rodent models of obesity exist, including diet-induced, monogenic, and polygenic models [76] . Because of the heterogeneity of obesity in humans, outbred models have been suggested as a better laboratory model for human disease [77] . Hibernating mammals, such as ground squirrels, represent a natural and outbred model for rapid adiposity and IR. These animals undergo a yearly cycle of fattening (preparing for hibernation) and weight loss (during hibernation), even on a calorie-restricted diet [78] . Hibernators also develop IR late in the active season only to become insulin sensitive again at the onset of hibernation [79] . Interestingly, the development of adiposity in ground squirrels mirrors the metabolic inflammation seen in obese rodent models and humans [80] . Thus, these animals serve as a good model in which to test the effects of manipulations of gut microbiota and inflammation as a treatment for adiposity and obesity. Hopefully, the use of outbred models more similar to humans in their heterogeneity of exposure, microbiota, and physiology can lead to better treatments for obesity that focus on curbing dysbiosis and metabolic inflammation.
Obesity is a heterogeneous disease, and treatments must be tailored to the individual patient. The pathway that we describe here represents a common etiology that arises from a changed diet and leads to the metabolic inflammation that is present in most cases of the disease. Changes to the gut microbiota and activation of the immune system are involved in many other diseases as well, including neurological disorders (reviewed in [81][82][83] ). It is important to develop a deeper understanding of the interactions between these important aspects of physiology to develop better treatments for a wide range of diseases in the future.

Conflicts of interest
The authors declare no conflicts of interest.

Funding
This work was supported by the National Institutes of Health award (2R15GM124586-02-"Microbiota and Inflammation in Adiposity: The Ground Squirrel Model").